Population‐level lateralization of boxing displays enhances fighting success in male Great Himalayan leaf‐nosed bats

Abstract Behavioral lateralization with left‐ and right‐hand use is common in the Animal Kingdom and can be advantageous for social species. The existence of a preferential use of the hands during agonistic interactions has been described for a number of invertebrate and vertebrate species. Bats compose the second largest order of mammals. They not only use their forelimbs for flight but also agonistic interactions. However, whether bat species show a population‐level lateralized aggressive display has largely been unexplored. Here, we examine the lateralization of boxing displays during agonistic interactions in male Great Himalayan leaf‐nosed bats, Hipposideros armiger, from three different populations. We found a population‐level lateralization of boxing displays: Males from all three populations show a preferential use of the left forearm to attack opponents. In addition, left‐handed boxers have higher fighting success over right‐handed boxers. This study expands our knowledge of the handedness of bats and highlights the role of behavioral lateralization in conflict resolution in nocturnal mammals.

their foraging behavior, fear responses, and aggressive behaviors (Rogers, 2002;Vallortigara & Rogers, 2005). Population-level biases can be evolutionarily stable as illustrated in the competitioncoordination model (Ghirlanda et al., 2009). This model predicts that minority-type individuals occur because they display unexpected fighting behaviors that rivals are less accustomed to during competitive interactions. Nonetheless, majority-type individuals gain a fitness advantage during interactions that require coordination, such as when mating. For example, minority-type giant Australian cuttlefish (Sepia apama) with a right-eye preference had higher success during fighting interactions, and majority-type individuals with a left-eye preference had higher success during mating interactions (Schnell et al., 2019).
To expand our understanding of behavioral bias in animal behavior, it is essential to explore the existence and the advantage of laterality during agonistic interactions in a broad range of vertebrates. Bats (Chiroptera) are unique mammals capable of flight and constitute the second largest radiation in mammals, with over 1456 species worldwide (Simmons & Cirranello, 2022). Echolocating bats not only emit echolocation pulses for navigation and prey acquisition but also communicate with a rich assortment of social vocalizations accompanied by behavioral displays (Chaverri et al., 2018). For example, many bats are involved in daily agonistic displays that function in territory defense or competition for mates. These displays included broadband calls, pushing, wing flapping, and boxing moves (Clement & Kanwal, 2012;Fernandez et al., 2014;Prat et al., 2016).
Here, we examine population-level lateralization in aggressive displays in the male Great Himalayan leaf-nosed bat, Hipposideros armiger, and test whether minority-type or majority-type lateralization enhances fighting success. H. armiger is a nocturnal and highly social species that usually roosts in caves, sharing day and night roosts among hundreds of individuals. Our previous studies showed that adult males defend their small, private roosting territory using conspicuous aggressive displays, e.g., boxing (Sun et al., 2019. Boxing is exhibited during escalated physical combat between two opponents, and males typically use only one forearm to knock each other until one of them retreats Zhang, Sun, Lucas, Gu, et al., 2022; Video S1). We hypothesized that a population-level lateralized boxing behavior would be displayed by male H. armiger. We predicted that there would be a significant difference in the proportion of a preferential use of the left or right forearms during agonistic interactions with a bias toward left-forearm use. If a population-level lateralized boxing behavior is detected, we further hypothesized that minority-type males would have an advantage in territorial conflict resolution based on the competition-coordination model. Therefore, we predicted that minority-type males will be more successful in fights than majoritytype males.

| Data collection
Two data sets were used in this study. For the first data set, we re- For the above two studies, each male was tested in only one agonistic interaction to avoid pseudoreplication. All of the agonistic interactions were monitored using night-shot camcorders (FDR-AX60, Sony Corp.). The experimental procedures are described in detail by Sun et al. (2019) and Zhang, Sun, Lucas, Gu, et al. (2022).

| Behavioral analysis
Agonistic interactions were analyzed using QvodPlayer software

| Morphological data
Body mass and forearm length of each experimental bat were obtained from Sun et al. (2019) and Zhang, Sun, Lucas, Gu, et al. (2022).
Individuals in each trial were size-matched with body mass values within 10% of each other and with the difference in the ratio of the forearm length divided by the average body length between the paired males <2% ± 1.2% Zhang, Sun, Lucas, Gu, et al., 2022).

| Statistical analyses
As noted above, data on bats from the Simao site were derived from two different studies (Sun et al., 2019;Zhang, Sun, Lucas, Gu, et al., 2022). We ran Pearson's chi-square tests to examine whether the proportion of hand preference and the proportion of laterality index significantly differed between Simao bats from the two studies. We found no significant differences in either parameter between bats from the two studies (Pearson's chi-square test: 2 1 = 0.070, p = .791; 2 1 = 0.682, p = .409, respectively). Therefore, we pooled these data sets for subsequent analysis.
We conducted the following statistical analysis for bats from Simao, Hekou and Hanzhong, respectively. Exact binomial probability tests were performed to compare the number of bats with a left-forearm and a right-forearm preference, and to compare the number of bats with a negative LI value and a positive LI value.
We also used kernel density estimates of the frequency distribution of laterality indices to offer a potentially more robust estimate of the entire frequency distribution. The kernel density estimation is a nonparametric method that adopts a slipped peak function to fit the sample data and utilizes a continuous density curve to describe the distribution pattern of the variables. It does not involve setting a functional form and can include observed variability in the data set with a continuous curve.
We also used an exact binomial probability test to evaluate whether the winners tend to display a left-forearm or a right-forearm preference. Moreover, in order to further confirm whether forearm preference has an effect on fighting ability, an exact binomial probability test was used to test whether the losers tend to display a left-forearm or a right-forearm preference.  Figure 1c).

| RE SULTS
Among the 119 fights, we only analyzed 20 interactions that involved size-matched opponents and involved boxing displays by both opponents. A majority of winners were more likely to exhibit a left-forearm preference than a right-forearm preference (binomial test: p = .041; Figure 1d). On the contrary, most losers had more right-forearm preferences than left-forearm preferences (binomial test: p = .041; Figure 1d).

| DISCUSS ION
In this study, we found that the proportion of left-handed boxers was significantly higher than the proportion of right-handed boxers, which supported our first hypothesis that male H. armiger show a population-level lateralized aggressive display. We also found that minority-type males (right-handed boxers) were less likely to succeed in fights than majority-type males (left-handed boxers), which failed to support our second hypothesis that minority-type males will play a dominant role in conflict resolution (Ghirlanda et al., 2009).
We found that there is a left-biased population-level lateralization of aggressive behaviors: Male H. armiger displayed boxing preferentially with the left forearm. This result is consistent with the evidence that behavioral lateralization at the population level is more likely to evolve in social species since it may provide an underlying social function to group-level behaviors (Deckel, 1995;. H. armiger is a highly social species, and frequent agonistic interactions among conspecifics during contests occurring in roosting space can be observed in day roosts (Sun et al., 2019;Yang, 2011). Population-level lateralized boxing displays in male H. armiger may serve as a component of resource-holding potential (Arnott & Elwood, 2009). Indeed, males with left-lateralized aggressive displays were more likely to win a contest.
Why did male H. armiger show a preferential use of the left forearm? One possible interpretation is that social animals may have a strong lateral preference to use left body parts due to the preeminence of the right hemisphere in aggressive interactions . There is also behavioral evidence that the presence of a right-side bias in the brain relative to the control of aggression is often linked to a preferential use of left body parts in aggressive interactions in a range of social animals including Mediterranean fruit flies, Ceratitis capitata (Benelli, Donati, et al., 2015), Australian stingless bees, Tetragonula carbonaria , red mason bees, Osmia bicornis , Magellanic penguins, Spheniscus magellanicus (Stor et al., 2019), and Przewalski horses, Equus przewalskii (Austin & Rogers, 2014).
Contrary to the prediction of the competition-coordination model, we found that minority-type males with a right-forearm preference were less likely to achieve fighting success. Similar results can be found in blowflies Calliphora vomitoria , Mediterranean fruit flies Ceratitis capitata (Benelli, Donati, et al., 2015), and olive fruit flies Bactrocera oleae . This result is not particularly surprising because male H.
armiger have been shown to make decisions in physical contests by assessments of their own ability (self-assessment) rather than of their combatant's relative ability (mutual assessment; Sun et al., 2019). Therefore, it is reasonable to suggest that the unexpected fighting behaviors with a right-forearm preference by an opponent would not affect an individual's decision to persist or to give up in a contest.
In summary, our study demonstrated that male H. armiger shows a population-level lateralized aggressive display with a left-forearm bias when using forearms for fighting. Moreover, left-handed boxers won more contests than right-handed boxers. To our knowledge, this is the first experimental evidence of lateralization of aggressive displays in a bat species. Further studies are needed to determine whether majority-type males (i.e., left-handed boxers) have a fitness advantage during intraspecific coordinated displays such as mating interactions.

ACK N OWLED G M ENTS
We would like to thank Hao Gu for field assistance.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no competing interests.